Fluorescent dyes are known to be particularly suitable for biological applications in which a highly sensitive detection reagent is desirable. Dyes that are able to preferentially bind to a specific biological ingredient in a sample enable the researcher to determine the presence or quantity of that specific ingredient. In addition, specific biomolecules can be monitored with respect to their spatial and temporal distribution in diverse environments. It is an object of this invention to describe fluorescent dyes that form a highly fluorescent complex with desirable spectral properties when combined with nucleic acids. It is a further object of this invention to describe the use of the fluorescent characteristics of the nucleic acid-dye complex to detect, identify, and quantify nucleic acids in a variety of media. It is an additional object of this invention to describe the detection, identification and quantification of nucleic acids when they are present in a variety of cells and cell types.
In many areas of basic research there is a need for rapid and sensitive detection of nucleic acids. Typically, this involves the analysis of complex mixtures of DNA, RNA, or nucleic acid fragments. In many fields of life sciences research, including biological, biomedical, genetic, fermentation, aquaculture, agricultural, forensic and environmental research, there is a need to identify nucleic acids both within and without cells as a routine component of standard experimental methods. A common example is the widespread use of gel electrophoresis for characterizing DNA, one limitation of which is the sensitivity of the staining method used to detect the faintest bands. Biological researchers and medical researchers often need to identify intracellular nucleic acids. Many scientists and medical technicians have a need to sort cells based on the amount of nucleic acid present in the cells. The amount of nucleic acid present in the cells can be indicative of the type of cells, or even the presence of disease states in cells (e.g. nucleated human red blood cells). Such applications require a fast, sensitive and selective methodology that can detect nucleic acids, even when bounded (or surrounded) by cellular membranes.
A generally applicable dye for staining nucleic acids preferably has the following properties:
1. The nucleic acid-dye complex should exhibit a very high signal with a low background, allowing the sensitive detection of minute quantities of nucleic acids, both cell-free and intracellularly.
2. A very high signal with a low background should be attainable with a low ratio of dye to nucleic acid. The small amount of dye binding to the nucleic acid should interfere as little as possible with the essential characteristics of the nucleic acids.
3. The nucleic acid/dye complex should exhibit photostability so that the fluorescent signal may be observed, monitored and recorded without significant photobleaching.
For specific applications involving live cells, additional necessary properties for a nucleic acid stain include:
4. The dye should be permeable to cell membranes so that it can bind nucleic acids sequestered in live cells.
5. The dye should require only brief incubation with the cells to obtain a detectable signal, and be relatively non-toxic to living cells, such that staining will not disrupt the normal metabolic processes of cells or result in premature cell death.
The dyes of the present invention have utility in any current application for detection of nucleic acids that requires a sensitive detection reagent. In particular, the dyes are useful for the detection of cell-free isolated nucleic acids, nucleic acids in solution, and nucleic acid bands in gels. Additionally, the present dyes greatly increase the sensitivity of detection of nucleic acids in a variety of cells and tissues, both living and dead, plant and animal, eukaryotic and prokaryotic. This family of dyes displays unusually good photostability and appear to be relatively non-toxic to cells. Furthermore, many of the dyes rapidly penetrate cell membranes of a variety of cells. The superior properties exhibited by these dyes were neither anticipated nor obvious in view of the known unsymmetrical cyanine dyes.
Although certain unsymmetrical cyanine dyes were first described before the genetic role of nucleic acids was established (Booker, et al., J. AM. CHEM. SOC. 64, 199 (1942)), a variety of unsymmetrical cyanine dyes have now been found to be very effective in the fluorescent staining of DNA and RNA. U.S. Pat. Nos. 4,554,546 (to Wang, et al. 1985) and 5,057,413 (to Terstappen et al. 1991) disclose use of similar thioflavin compounds as nucleic acid stains. The nondimeric unsymmetric cyanine dye sold under the tradename Thiazole Orange has particular advantages in the quantitative analysis of immature blood cells or reticulocytes (U.S. Pat. No. 4,883,867 to Lee, et al. (1989)) or in preferentially staining the nucleic acids of bloodborne parasites with little staining of nucleated blood cells (U.S. Pat. No. 4,937,198 to Lee et al. (1990). Although Thiazole Orange and other thioflavin cyanine dyes are permeant to many mammalian cells, these dyes are impermeant to some eukaryotic cells. Other related cyanine dye compounds are described in copending applications DIMERS OF UNSYMMETRICAL CYANINE DYES (Set. No. 07/761,177 to Yue, et al. filed Sep. 16, 1991) now abandoned, UNSYMMETRICAL CYANINE DYES WITH CATIONIC SIDE CHAIN (Ser No. 07/833,006 to Yue, et al. filed Feb. 8, 1992) now U.S. Pat. No. 5,321,130, and DIMERS OF UNSYMMETRICAL CYANINE DYES CONTAINING PYRIDINIUM MOIETIES (Ser. No. 08/043,665 to Yue, et al. filed Apr. 5, 1993) now U.S. Pat. No. 5,410,030. These dyes are non-permeant to living cells, unless the cell membrane has been disrupted.
The inventors have discovered that attachment of various cyclic structures to a parent unsymmetrical cyanine produces a hmily of superior nucleic acid dyes. Like other unsymmetrical cyanine dyes, this new family of dyes exhibits extremely low fluorescence in the absence of nucleic acids, and a large fluorescence enhancement when bound to nucleic acids. The compounds of the present invention, however, show certain advantages for nucleic acid detection with respect to the known cyanine dyes. Surprisingly, although bulkier, the new dyes more quickly penetrate the cell membranes of a wider variety of cell types, including both gram-positive and gram-negative bacteria, yeasts, and eukaryotic cells as well as prokaryotic cells. The subject dyes also more rapidly stain elcctrophoretic gels used for the separation of nucleic acids. Direct comparison of the rate of uptake in bacteria with known dyes such as Thiazole Orange and its homologs, shows enhanced uptake of the new compounds (Table 1). Moreover, bacteria stained with the unsymmetrical dyes with cyclic substituents may exhibit greater than tenfold more fluorescence than bacteria stained with Thiazole Orange (Table 2, normalized data in Table 3). Even in applications where cell permeability is not a factor, the quantum yield of most of these dyes is unexpectedly, and significantly, better than that of Thiazole Orange (Table 4). Furthermore, by simple synthetic modification, a family of dyes having absorption and emission spectral properties that cover most of the visible and near-infrared spectrum can be prepared. The improved fluorescent properties of the dyes of the present invention, in combination with nucleic acids, present significant advantages in all areas of nucleic acid detection.